The authors acknowledge funding sources from D&#8217;Youville College School of Pharmacy, Buffalo, NY.

1. Farklı Medya ve Ek Bileşenleri hazırlayın <ol><li> Deneyden önce, test edilecek da normal FBS ilave karakalem sıyrılmış FBS ya da kömür şeritli FBS artı madde ile DMEM veya diğer belirtilen ortam maddelerini kapsayan ortamı hazırlar. Birden fazla bileşen, her deneyde test edilebilir unutmayın. </li><li> Tartın ve uygun fizyolojik konsantrasyon tarafından kömür şeritli serum içinde çözünmüş hormonlar, büyüme faktörleri ya da sitokinler seyreltin. </li></ol> 2. on Ice Kollajen Matrix hazırlayın <ol><li> 200 ul 10x PBS (pH 7.4), 5.4 ul 1 N NaOH, iki kez damıtılmış H2O 600 ul ve 1.2 ml kolajen I: buzda sonra steril filtre edilmiş bileşenler eklenerek 2.2 mg / ml&#39;lik bir matris 2 mi kolajen hazırlanması (de 3.63 mg / ml). </li><li> Plaka için hazır olana kadar buz üzerinde kolajen I çözeltisi tutun. </li></ol> 3. &lt;Testi için Göç / Invasion Tabaklar hazırlayın/ P&gt; <ol><li> Her bir hücre çizgisi test edilebilmesi için, 12-kuyu ekler ihtiva eden bir 24-çukurlu plaka bölmesi kullanırlar. Kemoatraktan medya ekleyerek ve deney düzeneği up için ekler aktarmak için ekler içermeyen ek 12 kuyu kullanın. NOT: hücre hatları burada kullanmak için bir polietilen tereftalat (PET) membran ve 8 mikron gözenek büyüklüğüne sahip plakalar üzerinde bir kollajen matriks uygunudur. Bununla birlikte, önceden kaplanmış plakalar içinde bir Matrigel matris, hücre çizgisi araştırılmaktadır göre azalmış gözenek boyutu ile ikame edilebilir. </li><li> Açıkça (bir kontrol, invaziv olmayan hücre hattı için FBS göç, CS-FBS göç, FBS işgali, ve CS-FBS işgali yanı sıra FBS-göç) analiz edilen duruma başına 3 kuyu kullanılarak, plaka etiket. Bir kolajen ya da Matrigel matris boyunca ve daha sonra zar içindeki gözeneklerin içinden hareket etmek sureti ile PET zar içindeki gözeneklerin içinden hareketi ve deney invazyonu ile deney göç. Bir her hücre durum için farklı renk işaretçileri kullanınKaplama işleminde kimliği. </li></ol> 4. Invasion Matrix dağıtın <ol><li> Dikkatle uçlar haline kollajen matris solüsyon içerisinde 75 ul istila deneyleri için kullanılacak pipetle. Kabarcıkları önlemek için dikkatli kullanın. Yüzeye ters pipet uygulayarak kabarcıklar Dağılın. </li><li> Katılaşmaya jel sağlamak için 30 dakika boyunca 37 ° C ve% 5 CO2 inkübatöründe kolajen kaplı insertler ile plaka aktarın. </li></ol> 5. Plaka Membran veya Invasion Matrix üzerine Hücreler <ol><li> Bu arada, Hücreleri tripsinize edin ve% 10 FBS ile ortamı eklenir. Sıkma hücreleri masa üstü bir santrifüj ile 5 dakika süreyle 200 x g&#39;de ve serumsuz ortamında 3 kez yıkayın. </li><li> Serum serbest medya süspanse. Bir hemasitometre veya otomatik slayt sayacı ile hücreleri saymak. 5 x 10 4 hücre / ml&#39;lik nihai bir konsantrasyona kadar serumsuz ortam ekleyin. </li><li> Kolajen matrisi (30 dakika sonra) katılaşmış girdiğinde, ortam 700 ul ya da2% tanımlanan FBS veya her kuyuya FBS kömür-elimden. Plaka başına 12 ekler: <ul><li> 3 ekler medya ile kolajen ve kuyuları var +% 2 FBS </li><li> 3 ekler medya ile kolajen ve kuyuları var +% 2 CS-FBS </li><li> 3 ekler medya +% 2 FBS ile hiçbir kolajen ve kuyuları var </li><li> 3 ekler medya ile hiçbir kolajen ve kuyuları var +% 2 CS-FBS </li></ul><ol><li> Test edilen faktörlerin sayısı ve her bir durumda karşılık gelen herhangi bir kollajen kontrol olarak, ek plakalar kullanın. </li></ol></li><li> Tüm geçiş ve istila insertlerin de uç başına 500 ul hücre vererek, 5 x 10 4 hücre / ml&#39;de ekler hücre süspansiyonu ekleyin. </li><li> 37 ° C&#39;de 22 saat boyunca kuluçkaya bırakılır. </li></ol> 6. Geçirme ve istila Hücrelerinin sayısı niceliğini <ol><li> Ayrı kuyularda metanol fiksatif, eozin ve hemotoksilin kullanarak kuyuların ayarlayın boyama. </li><li> Her kuyudan hücreleri ve matriks çıkarmak için pamuklu bezlerden kullanın.Her kuyu için ikinci sürüntü uygulaması ile Rrepeat. </li><li> Forseps ile, arka arkaya 3 çözeltilerin her birine, her bir ek 1 saniye için 5 kez batırın. </li><li> Ekler / N O kurumasını bekleyin. </li><li> Ya i) kenarlarda dikkatle kesim, bir neşter ile filtreleri kaldırabilirsiniz ve lamel ve daldırma yağ ile bir slayt monte, veya ii) ekler O / N ters kurumasını ve mikroskopi için doğrudan ekler kullanmak için izin. </li><li> Ertesi gün, bir 20X amacı ile bir mikroskop altında slaytlar veya ekler görüntüleyebilir ve filtrenin farklı bölgelerinden 5 görüntüleri çekmek. Tutarlılığı artırmak için, 4 dış alanları ve bir merkez almak. </li><li> ImageJ yazılımı kullanarak tüm koşullar için hücreleri saymak ve aşağıdaki formüller için geçerlidir. </li><li> Aşağıdaki gibi yüzde işgali belirleyin: Ben = a eklemek kollajen sayesinde işgalci hücrelerin # ortalama Kontrol eki aracılığıyla göç hücrelerin # = b Ortalama % Işgali = (a / b) x 100 </li><li> Aşağıdaki gibi% 2 FBS Invasion Endeksi belirleyin: Hücrelerin istilası% (% 2 FBS içinde) analiz edilir = C (% 2% FBS) kontrol girişimsel olmayan hücrelerin işgali = d Invasion İndeksi (FBS) = (c / d) </li><li> Aşağıdaki gibi% 2 CS-FBS Invasion Endeksi belirleyin: Hücrelerin istilası% = e (% 2 CS-FBS) analiz edilir % (% 2 CS-FBS) kontrol girişimsel olmayan hücrelerin işgali = f Invasion Endeksi (CS-FBS) = (e / f) </li></ol> 7. tekrarlayın Deneysel Protokolü + X n Çoklu Faktörler Kombine ile Kömür-elimden FBS ile FBS Kömür-elimden Karşılaştırılması <ol><li> &quot;X&quot; ya da bileşenlerin bir kombinasyonu, farklı bileşenler kullanılarak gerektiği kadar prosedür birden fazla kez tekrarlayın. </li><li> Göç ve istila etkileri her faktör &quot;X&quot; katkısını belirlemek için hesaplamalar uygulayın. </li></ol>

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The authors have nothing to disclose.

Tümör metastaz çok aşamalı bir süreçtir. Hücreler, bazal membran kırmaya da uzak bölgelere taşınabilen lenf sistemi veya kan ya da kılcal damarlarla içine intravasate gerekir. Tümör hücreleri daha sonra damar dışına ve Makrometastaz 12 içine yerleşmektedir. Mezenkimal geçiş (EMT), tümör invazyonu ve metastaz için epitel yoluyla ilerleme steroid hormonların 13,14 tarafından geliştirilmiştir, büyüme 15-18, ve sitokinler 19-21 faktörleri. Bu mole...

Bir yeni işgal tahlil belirli hormonlar, kanser hücresi yayılımını tamamen içinde kimyasal çekici olarak sitokin ve / veya büyüme faktörlerinin katılımı belirlenmesi amacı ile geliştirilmiştir. hücre dışı matriks (ECM) ile işgal etmeye tümör hücrelerinin yeteneği metastatik fenotip 1-3 bir özelliğidir. İstila odaları yaygın olarak kanser hücresi istilasını ve göç çalışma, in vitro araç olarak kullanılmıştır ve in vivo tümör istilası ve metastaz mekanizmaları da bilgiyi sağlayabilir. odalar, hücre kültür plakalarının gözeneklerine içinde iç içe geçmiş silindir hücre kültürü ekler oluşur. insertlerin taban, tanımlanmış gözenek büyüklüğü ve yarı geçirgen polikarbonat veya polistirol membranlardır. Standart istila deneyinde, hücreler, serumsuz ortam ile uç zar üzerine ekilir ve serum veya serum gibi kemoatraktantlarla dolu olan hücre kültür çukurlarına yerleştirildi set kadar kemoatraktif kuvvet. Bu kuvvet, tahrik Hücreler daha sonra tespit etmek ve hücrelerin sayısını belirlemek için geleneksel boyaları kullanılarak boyanmış hücre dışı matris (yayılma) &#39;in bir kaplama boyunca, alternatif olarak, yarı geçirgen bir zar (göç) hareket ettirin. hücre sayısı daha sonra invaziv olmayan hücre hattında göç ve istila derecesine göre normalize edilir. Bu yöntem araştırmacının, genetik manipülasyon da dahil olmak üzere koşullar altında çeşitli farklı hücre tiplerinin invaziv potansiyelini değerlendirmek için izin verir. Hormonlar, sitokinler ve büyüme faktörleri serum kritik bileşenleridir ve giderek invaziv fenotip 4 sürüş ile ilişkili olduğu gösterilmiştir edilmektedir. Ancak, yine de işgali aracılık bu kemoatraktif serum moleküllerinin yapısı, rolü ve özgüllük zor kalır. meydan sürüş sorumlu serum veya serum benzeri chemoattractants belirli hangi faktörlerin belirlenmesi kalırmoleküller işgali sürecini inhibe ne olabilir kanser hücresi işgali yanı sıra. Bu yazıda, kanser hücrelerinin chemoattraction ve işgali sürüş molekülleri olarak, hormonlar, sitokinler ve / veya serum içinde mevcut olan büyüme faktörleri potansiyel katılımı değerlendirmek için bir yöntem tarif eder. Önerilen bu tadil edilmiş protokolde, odun kömürü sıyırma% 2 karakalem sıyrılmış FBS (CS-FBS) oluşturmak üzere,% 2 fetal sığır serumu (FBS) ile ilgili hormonlar, sitokinler ve büyüme faktörleri çıkarmak için kullanılır. % 2 FBS ve% 2 CS-FBS iki kanser hücre istilasını sürücü kemoatraktif kuvveti ayarlamak için maddeler olarak kullanılmaktadır. Normal% 2 FBS ile karşılaştırıldığında, kemoatraktif madde olarak% 2 CS-FBS kullanarak ilk dahil olmak üzere çeşitli yararlar elde edilir, ve en belirgin olarak, hormonlar, sitokinler ve büyüme faktörleri, indirgenmiş / bağıl yokluğunda kanser istilası fenotipi çalışma yeteneği. Ayrıca hormonlar, büyüme faktörleri kolektif çıkarılması olup olmadığını değerlendirmek için araştırmacı sağlar ve cytokines invaziv endeksteki artış veya azalmaya neden olur. Daha sonra deney, (Şekil 2 ve 3 e bakınız), &quot;X&quot; olarak adlandırılan tek bir bileşen eklenmesi azalma artışı önleyen, ya da orijinal değerine invaziv endeksi geri olup olmadığını belirlemek için tasarlanmıştır. Bu yöntem, kömür sıyırma boyunca serum uzaklaştırılır bileşen fizyolojik seviyelerini elde etmek için spesifik olan, ancak, aynı zamanda kömür sıyırma aynı zamanda sinyal iletim yollarını etkileyen unutulmamalıdır. Örneğin, odun kömürü sıyırma osteoprogenitör hücrelerinin alkalin fosfataz aktivitesi azalır ve MAPK nin aktivatörlerinin 5 azaltılması yoluyla adipogenesis uyarabilir bildirilmiştir. Karakalem sıyrılmış ortam ticari olarak temin edilebilir ve protokolüne göre bu dekstran ile kömür birleştirir ve fetus inek serumu, O / N 6 ile inkübe özetlenmiştir. Bu deney, RESPO geliştirilenNSE Zucker ve ark. 7 tarafından bildirilen bir sonuç yazarlar, normal% 2 FBS ile medya doğru göç sırasında gap junction iletişimi etkileyen bir mutant fenotip işgali endeksindeki artış gösterdi olduğunu gösterdi. Bu artış, kömürle sıyrılmış serum ikamesi ile ortadan kaldırıldı. Bu sonuçtan, Yazarlar, bir lipofilik molekül yönelik bu mutan etkilenen taşıma (daha özel olarak ise, bir hormon, bir büyüme faktörü, sitokin ya da) 7. İyi hormonlar, büyüme faktörleri ve sitokinler tümör promosyonuyla ve istila 4 yer alan sinyal yolları aracılık ettiği bilinmektedir. Bu nedenle, bu bilim araştırmacılar ne faktör (ler) çalışma altında, özellikle kanser hücrelerinin veya mutantlar tümör invazyonu sürüş belirlemek için de önemlidir. bileşenin konsantrasyonu arasındaki farka bağlı olarak, belirlenen deney fizyolojik konsantrasyonda ayrı ayrı bileşenlerin rolünü ele almak üzere tasarlanmıştır ve# 8220, X &quot;, normal FBS ve CS-FBS içinde. Bu testin gelişme sayesinde, araştırmacılar kendi sistemini düzenleyen özel invaziv yolun içine daha fazla fikir sahibi olabilir. Hormonlar, büyüme faktörleri, sitokinler ve genellikle tümör destekleyicileri 8 olarak sınıflandırılmıştır. Örneğin EGF, bu faktörlerden bazıları istila odaları 9 kimyasal çekici doğrudan kaynakları olarak kullanılmaktadır. Bu nedenle, bu doğrudan tümör invazyonu bu serum ana bileşenleri temsil gibi görünmektedir. Bu protokol bir araştırmacı kanser hücrelerinin invaziv potansiyeli aracılık hormonlar, büyüme faktörleri ve sitokinler katılımını değerlendirmek için izin verir geleneksel in vitro işgali tahlil için basit, ama önemli, değişiklik önermektedir. Ancak, tahlil değerli zaman ve r kullanmak şekilde değil, prosedür analizinde erken aşamada yaptıkları çalışmada etkili olacaktır dair bir cevap ile araştırmacı sağlamak için tasarlanmıştıresources gereksiz görülmesi halinde. Bu yöntem CS-FBS kullanır ve potansiyel kanser hücresi işgali aracılık kurşun adayları olarak sürdürmeye molekülleri hangi şekilde araştırmacılar takdirine bağlıdır. Bu analizlerin sonuçları, serum bileşenleri, özellikle hücre hattı veya mutant için Kemoattraktantlar veya inhibitörleri çalışılan olarak hizmet hangi belirlenmesinde yararlı olduğu ispatlamak zorundadır. Ayrıca, bu yaklaşım araştırmacı teşvik veya kanser hücresi işgali inhibe ya anahtar sinyal yolları belirlemeye yardımcı olabilir; Böylece gelecekte ilaç tasarımı yönlendiren.

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:367px;">Item</td>
			<td style="width:195px;">Vendor</td>
			<td style="width:136px;">Catalog #</td>
			<td style="width:289px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td>BioCoat Control Inserts with 8.0&#181;m PET Membrane 
			(12 in each of two 24-Well Plates)</td>
			<td>Corning</td>
			<td>354578</td>
			<td>polyethylene teraphthalate (PET) membranes&#160;&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Collagen I, rat tail</td>
			<td>Corning</td>
			<td>354236</td>
			<td>Source = rat tail tendon, purity = 90%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fixative; Diff-Quick</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9844047</td>
			<td>Methanol-based fixative, Hmatoxylin/Eosin</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal bovine Serum</td>
			<td>Life Technologies</td>
			<td>16000</td>
			<td>Designated as FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Charcoal Stripped Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>12676</td>
			<td>Designated as CS-FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal bovine Serum</td>
			<td>Thermo Scientific Hyclone</td>
			<td>SH30070</td>
			<td>Designated as FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Charcoal Stripped Fetal Bovine Serum</td>
			<td>Thermo Scientific Hyclone</td>
			<td>SH30068</td>
			<td>Designated as CS-FBS</td>
		</tr>
	</tbody>
</table>

a modified in vitro invasion assay to determine the potential role of hormones, cytokines and/or growth factors in mediating cancer cell invasion

Kan serumu kanser hücrelerinin mikrodamarlara kendi intravasation kolaylaştırılması, göç ve istila hangi doğru kemoatraktan olarak hizmet vermektedir. Ancak, hücre göç hangi doğru gerçek moleküller zor kalır. Bu modifiye işgali tahlil hücre göçü ve işgali sürücü hedefleri belirlemek için geliştirilmiştir. Bu teknik, belirli bir hormon, büyüme faktörü, sitokin ya da bir kanser hücresinin invaziv potansiyel aracılık bir rol oynayıp oynamadığını belirlemek için üç koşullar altında istila endeksi karşılaştırır. Bu koşullar, i), normal fetal sığır serumu (FBS), ii) kömür-sıyrılmış hormonlar, büyüme faktörleri ve sitokinler ve iii) CS-FBS + molekülü kaldırır FBS (CS-FBS) (&quot;X&quot;) gösterilir içerir. FBS ile karşılaştırıldığında CS-FBS ile hücresi yayılmasında belirgin bir değişiklik, değişiklik aracılık hormonlar, sitokinler ya da büyüme faktörleri olduğu anlamına gelir. Bağımsız molekülleri daha sonra yeniden kabiliyetlerini test etmek için, CS-FBS geri eklenebilirayet ya da işgali fenotip kurtarmak. Ayrıca, iki ya da daha çok faktör katkı maddesi veya araç veya yayılmasının inhibe edilmesinde çok sayıda molekülün sinerjistik etkilerini değerlendirmek için kombine edilebilir. Genel olarak, bu yöntem, hormonlar, sitokinler ve / veya büyüme faktörlerinin kanser hücresinin özel bir tip ya da belirli bir mutant için kimyasal çekici ve istila inhibitörleri olarak görev yapan hücre istilasında rol oynayan olup olmadığını belirlemek için araştırmacı sağlar. Belirli chemoattractants ve inhibitörleri tanımlayarak, bu değiştirilmiş işgali tahlil kanser hücresi işgali yönlendiren sinyal yolları aydınlatmak için yardımcı olabilir.

işgal endeksi noninvaziv hücre hattına normalleşme göre her durum için hesaplanır. Deneyler için, biz 1205Lu melanom hücre çizgisi ve kurulan varyant kararlı hücre kuşaklarının z invaziv çizgiler olarak hem de 1205Lu hücreleri mantıksal kontrol olarak hizmet vermektedir 10 türetildikleri premalign invaziv olmayan değişik türde, WM793 kullanın. Bu dermis birincil bileşeni olduğundan biz de işgali matrisi olarak kollajen I kullanmaktadır. Optimal işgali matris hücre hattı dayalı v...

Watch this Scientific Journal Video about A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion at JoVE.com

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Bir Modifiye<em&gt; In vitro</em&gt; Invasion Deneyi Hormonlar, Açan Kanser Hücre Invasion Sitokinlerin ve / veya Büyüme Faktörleri Potansiyel Rolü belirlemek için

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

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A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

10.6K Görüntüleme.  Roswell Park Cancer Institute. This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Video: A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

Kanser Hücreleri Endotel Hücre Tek tabakalı Invasion Tedbir Gerçek zamanlı Elektrik Empedans Tabanlı Tekniği

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

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An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Bir In Vitro</emMetastatik Büyüme Tümör Dormansi ve Switch Eğitim> Sistem

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Oral Skuamöz Hücre Karsinomu Ortotopik Fare Modeli Tümör Hücre Invasion Multi-foton Görüntüleme

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF-β-kaynaklı Invasion Tedbir Sfero Testi

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modelleme ve Görüntüleme 3-Boyutlu Toplu Hücre Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

In vitro Mezotelyal Gümrükleme Assay o Yumurtalık Kanseri Metastaz Erken Adımlar modeller

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Real Time Mezotelyal Hücrelerinin Yumurtalık Kanseri Sferoid Ek ve Invasion Değerlendirilmesi

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Formation Odak: bir hücreye dayalı tahlil, bir genin onkojenik potansiyeli belirlemek için

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Chicken İnsan Multipl Miyelom Xenograftlarında Modeli Kurulması Tümör Büyüme, Invasion ve Anjiogenez Eğitim için

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Üç boyutlu (3D) Tümör Sfero Yayılması deneyindeki

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...